Scaffolded HIV-1 vaccine immunogens

ABSTRACT

The present invention provides novel scaffolded HIV-1 vaccine immunogens. Some of the scaffolded immunogens contain a soluble gp140 trimer linked to the N-terminus of the nanoparticle subunit and a T-helper epitope that is fused via a short peptide spacer to the C-terminus of the nanoparticle subunit. Some other immunogens of the invention contain a soluble gp140 trimer protein that is linked to a stable nanoparticle via a short peptide spacer that is a T-helper epitope. Some of the scaffolded immunogens contain a gp140 trimer immunogen presented on a nanoparticle platform formed with I3-01 protein, E2p, or variants of protein 1VLW. Also provided in the invention are nucleic acids that encode the various vaccine immunogens described herein, and expression vectors and host cells harboring the nucleic acids. The invention further provides methods of using the scaffolded HIV-1 vaccine immunogens for preventing or treating HIV infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject patent application claims the benefit of priority to U.S.Provisional Patent Application No. 62/580,038 (filed Nov. 1, 2017; nowpending). The full disclosure of the priority application isincorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI100663, A1084817, GM094586 and AI110657 awarded by The NationalInstitutes of Health and grant number DE-AC02-76SF00515 awarded by theU.S. Department of Energy. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type-1 (HIV-1) is the primary cause of theacquired immune deficiency syndrome (AIDS). It can be divided intoseveral different clades, for example A, B, C, D, E, F, G, H, J and K,which vary in prevalence throughout the world. Each clade comprisesdifferent strains of HIV-1 which have been grouped together on the basisof their genetic similarity. The envelope glycoprotein (Env) of HIV-1harbors the epitopes of broadly neutralizing antibodies (bNAbs) and isthe sole target of vaccine design. The cleaved, mature Env is presentedon the HIV-1 virion surface as a metastable trimer of heterodimers eachcontaining a (co-) receptor-binding protein, gp120, and a transmembraneprotein, gp41, which anchors the trimeric spike in viral membrane anddrives the fusion process during cell. Due to the labile nature and adense layer of surface glycans, Env has long resisted structuredetermination and hampered trimer-based vaccine efforts.

Native-like Env trimers have recently been considered a desirablevaccine platform due to the promising successes achieved with the BG505SOSIP.664 trimer. In addition to SOSIP, other trimer design platformssuch as the single-chain gp140 (sc-gp140) trimer, native flexibly linked(NFL) trimer, and uncleaved prefusion-optimized (UFO) trimer were alsoproposed that produced native-like Env trimers. However, gp140 trimermay not be the optimal form of an HIV-I vaccine because subunit vaccinesare often less immunogenic than virus-like particles (VLPs), whichpresent a dense array of antigens on the particle surface and inducepotent, long-lasting immune responses upon vaccination.

Despite the increasing appreciation for the advantages of VLP vaccinesin bNAb elicitation, the utility of nanoparticles as carriers to displaynative-like trimers has not been rigorously explored in HIV-1 vaccinedevelopment. There remains to be an unmet medical need for safe andefficacious HIV-1 vaccines. The present invention addresses this andother needs in the art.

SUMMARY OF THE INVENTION

In one aspect, the invention provides HIV-1 vaccine immunogens. Thenovel HIV-1 vaccine immunogens of the invention contain an HIV-1Env-derived trimer protein presented on a self-assembling nanoparticleand also a T-helper epitope sequence. In some embodiments, the T-helperepitope links C-terminus of the HIV-1 trimer protein subunit to theN-terminus of the nanoparticle subunit. In some other embodiments, theT-helper epitope sequence is fused to the C-terminus of the nanoparticlesubunit while C-terminus of the HIV-1 trimer protein is fused to theN-terminus of the nanoparticle subunit. In some of the latterembodiments, a short peptide spacer is used to fuse the T-helper epitopeto the nanoparticle subunit. This allows formation of a hydrophobic coreinside the nanoparticle, which functions to stabilize the nanoparticlestructure and to promote T cell recognition of the fusion immunogen. Insome of these embodiments, the employed short peptide spacer can be,e.g., 1-5 tandem repeats of GGGGS (SEQ ID NO:4) or GSGSG (SEQ ID NO:19),or any other peptide sequence that is structurally flexible by nature.In some of these embodiments, an additional short peptide segment orspacer can be used to fuse the HIV-1 protein to the N-terminus of thenanoparticle subunit, e.g., a 1G linker or any of the other shortpeptide spacers described herein. In some embodiments, the T-helperepitope sequence contains an amino acid sequence as shown in any one ofSEQ ID NOs:1-3, a conservatively modified variant or a substantiallyidentical sequence thereof.

Typically, the self-assembling nanoparticle in the HIV-1 vaccineimmunogens is generated with a trimeric protein sequence. In someembodiments, the subunit of the self-assembling nanoparticle is (1) thepolypeptide as shown in SEQ ID NO:18, a conservatively modified variantor a substantially identical sequence thereof, (2) the polypeptide asshown in any one of SEQ ID NOs:5-17, a conservatively modified variantor a substantially identical sequence thereof, (3) E2p or (4) ferritin.

In various embodiments, the HIV-1 Env-derived trimer protein in thevaccine immunogens of the invention is a gp140 trimer. In someembodiments, the employed HIV-1 Env-derived trimer protein is anuncleaved prefusion-optimized (UFO) gp140 trimer. In some of theseembodiments, the UFO gp140 trimer is a chimeric trimer comprising amodified gp41_(ECTO) domain from HIV-1 strain BG505. Some HIV-1 vaccineimmunogens of the invention contain a HIV-1 Env-derived trimer that isan UFO gp140 trimer, a self-assembling nanoparticle that is generatedwith a subunit sequence as shown in any one of SEQ ID NOs:5-18, and aT-helper epitope that contains the sequence as shown in SEQ ID NO:1.

In another aspect, the invention provides HIV-1 vaccine immunogens thatcontain an HIV-1 Env-derived trimer protein presented on aself-assembling nanoparticle that is formed with a subunit polypeptideas shown in any one of SEQ ID NOs:5-18, a conservatively modifiedvariant or a substantially identical sequence thereof. In someembodiments, the employed HIV-1 Env-derived trimer protein is anuncleaved prefusion-optimized (UFO) gp140 trimer. In some of theseembodiments, the UFO gp140 trimer is a chimeric trimer that contains amodified gp41_(ECTO) domain from HIV-1 strain BG505. In someembodiments, the HIV-1 trimer protein in the HIV-1 vaccine immunogen islinked at its C-terminus to the N-terminus of the nanoparticle via alinker sequence. In some other embodiments, the linker sequence is fusedto the C-terminus of the nanoparticle subunit via a short peptide spacerto form a hydrophobic core inside the nanoparticle while the UFO gp140trimer subunit is fused to the N-terminus of the nanoparticle subunit.This functions to stabilize the nanoparticle structure and to promote Tcell recognition of the trimer immunogen. In some of these embodiments,the employed short peptide spacer can be, e.g., GGGGS (SEQ ID NO:4),GSGSG (SEQ ID NO:19), or any other peptide that is structurally flexibleby nature. In various embodiments, the employed linker sequence containsa T-helper epitope sequence or a glycine-serine linker. In someembodiments, the linker sequence contains the peptide sequence as shownin any one of SEQ ID NOs:1-3, a conservatively modified variant or asubstantially identical sequence thereof. In some embodiments, thelinker sequence contains 1 to 5 tandem repeats (e.g., 1 or 2 repeats) ofGGGGS (SEQ ID NO:4) or GSGSG (SEQ ID NO:19). In some embodiments, anadditional short peptide spacer or segment can be used to fuse the HIV-1protein to the N-terminus of the nanoparticle subunit as exemplifiedherein.

In a related aspect, the invention provides pharmaceutical compositionsthat contain one of the novel scaffolded HIV-1 vaccine immunogensdescribed herein. The pharmaceutical compositions typically also containa pharmaceutically acceptable carrier. In some embodiments, thepharmaceutical compositions additionally contain an adjuvant. In anotherrelated aspect, the invention provides isolated or recombinantpolynucleotides that encode the HIV-1 vaccine immunogens describedherein, cloning and expression vectors harboring such polynucleotidesequences, as well as host cells into which the nucleic acids or vectorshave been introduced or integrated.

In another aspect, the invention provides methods for preventing HIV-1infection or eliciting an immune response against HIV-1 in a subject.These methods entail administering to the subject a therapeuticallyeffective amount of one of the novel scaffolded HIV-1 vaccine immunogensdescribed herein. Typically, the HIV-1 vaccine immunogen is administeredto the subject via a pharmaceutical composition. In some embodiments,the administered HIV-1 vaccine immunogen contains an UFO gp140 trimer, aself-assembling nanoparticle generated with a subunit sequence as shownin SEQ ID NO:18, and a T-helper epitope sequence as shown in SEQ IDNO:1. In these embodiments, the T-helper epitope sequence functions tocovalently link the UFO gp140 trimer at its C-terminus to the N-terminusof the nanoparticle subunit. Alternatively, the T-helper epitopesequence is fused to the C-terminus of the nanoparticle subunit via ashort peptide spacer while the UFO gp140 trimer subunit is fused to theN-terminus of the nanoparticle subunit.

In another aspect, the invention provides methods for treating HIV-1infection or eliciting an immune response against HIV-1 in a subject.The methods involve administering to the subject a pharmaceuticalcomposition that contains a therapeutically effective amount of a HIV-1vaccine immunogen described herein. In some embodiments, theadministered HIV-1 vaccine immunogen contains an UFO gp140 trimer, aself-assembling nanoparticle generated with a subunit sequence as shownin SEQ ID NO:18, and a T-helper epitope sequence as shown in SEQ IDNO:1. In these methods, the T-helper epitope sequence functions tocovalently link the UFO gp140 trimer at its C-terminus to the N-terminusof the nanoparticle subunit. Alternatively, the T-helper epitopesequence is fused to the C-terminus of the nanoparticle subunit via ashort peptide spacer while the UFO gp140 trimer subunit is fused to theN-terminus of the nanoparticle subunit.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ferritin nanoparticles presenting diverse UFO²-BG trimersand I3-01-based gp140 nanoparticles with embedded T-help signal. (A)Surface model of UFO²-BG gp140-ferritin (FR) nanoparticle, with gp120,BG505 gp41_(ECTO) of the UFO design, and ferritin circled by dottedlines on the gp140-FR images and labeled with arrows. (B) BN-PAGE ofeight UFO²-BG-FR nanoparticles after a single-step 2G12 antibodyaffinity purification. (C) Reference-free 2D class averages derived fromnegative-stain EM of five representative UFO²-BG-FR nanoparticles. (D)Antigenic profiles of five representative UFO²-BG-FR nanoparticlesagainst a small panel of six bNAbs and four non-NAbs. Sensorgrams wereobtained from an Octet RED96 using an antigen titration series of sixconcentrations (starting at 35 nM by two-fold dilution). The peak valuesat the highest concentration are summarized in the matrix, in which sixbNAbs and four non-NAbs are shown in upper and lower panels,respectively. Higher intensity of gray shade indicates greater bindingsignal measured by Octet. (E) Surface model of the I3-01 nanoparticle(light gray) is shown on the left, with the subunits surrounding afront-facing 5-fold axis highlighted in dark gray and three subunitsforming a 3-fold axis marked with a black dotted-line triangle. Thespacing between N-termini of three I3-01 subunits surrounding a 3-foldaxis (top view) and the anchoring of a gp140 trimer onto three I3-01subunits by flexible linkers (indicated by black dotted lines) are shownin the middle. Schematic representation of I3-01 nanoparticle constructscontaining both gp140 and a T-helper epitope is shown on the right, withsequences listed for three such T-helper epitopes, PADRE, D, and TpD(SEQ ID NOs:1-3, respectively). (F) SEC profiles of three I3-01nanoparticles presenting an HR1-redesigned BG505 gp140 trimer withdifferent T-helper epitopes as linkers. (G) BN-PAGE of threeabovementioned I3-01 nanoparticles after a single-step 2G12 affinityantibody purification. (H) Reference-free 2D class averages derived fromnegative-stain EM of an I3-01 nanoparticle presenting an HR1-redesignedBG505 gp140 trimer with PADRE used as a linker. (I) Antigenic profilesof gp140-PADRE-I3-01 nanoparticle against a small panel of six bNAbs andfour non-NAbs. Sensorgrams were obtained from an Octet RED96 using anantigen titration series of six concentrations (starting at 14 nM bytwo-fold dilution). The six antigen concentrations, respectivelycorresponding to the six lines from top to bottom in each of the 10antibody profiles, are indicated next to the PGT151 binding profile onthe right.

FIG. 2 shows effective B cell activation by trimer-presentingnanoparticles. Ca²⁺ mobilization in B cell transfectants carrying (A)PGT145, (B) PGT121, and (C) VRC01 receptors. WEHI231 cells expressing adoxycyclin-inducible form of bNAb B cell receptor (BCR) were stimulatedwith anti-BCR antibodies or the indicated antigens at a concentration of10 μg ml⁻¹: anti-human Ig κ-chain F(ab′)2; anti-mouse IgM; an UFO²-BG-FRnanoparticle derived from a clade-A, B, C, B/C, or A/E strain, or BG505gp140-PADRE-I3-01 nanoparticle containing a redesigned HR1 bend withingp41_(ECTO).

FIG. 3 shows early neutralizing antibody responses to trimers andnanoparticles in mouse immunization. (A) Schematic view of mouseimmunization protocol is shown on the left, with the key parameters offormulation and immunization listed in the middle, and serum IgGpurification protocol on the right. (B) Testing BG505 trimer-basedimmunogens and ELISA binding of purified mouse serum IgGs to three HIV-1antigens, including BG505 UFO trimer, a ferritin nanoparticle presentingan N332 scaffold (1GUT_A_ES-FR) or an I3-01 nanoparticle presentinganother N332 scaffold (1KIG L ES-2-I3-01), and a clade-C V1V2-ferritinnanoparticle (V1V2-FR). EC₅₀ values are labeled for all ELISA plotsexcept for instances in which the highest OD₄₅₀ value is below 0.1 or inthe cases of ambiguous data fitting. (C) HIV-1 neutralization bypurified mouse serum IgG, with IC₅₀ values shown in gray shade. Higherintensity of gray shade indicates more potent neutralization. (D)Neutralization profile of group-combined mouse serum IgG from thescaffolded trimer group (S1G5). (E) Neutralization profile ofgroup-combined mouse serum IgG and mouse-A serum IgG from the ferritinnanoparticle group (S2G1). (F) Neutralization profile of group-combinedmouse serum IgG, mouse-A and mouse-D serum IgG from the I3-01nanoparticle group (S2G5). Two HIV-1 pseudoviruses, clade-A tier-2 BG505and clade-B tier-1 SF162, were tested with MLV included for comparison.The structural models of scaffolded gp140 trimer, ferritin nanoparticle,and I3-01 nanoparticle are shown next to the neutralization profile ofgroup-combined mouse serum IgG.

FIG. 4 shows the design concept, SEC profile, and negative-stain EMimage of HIV-1 gp140 trimer-presenting nanoparticle with a T-helperepitope fused to the C-terminus of the nanoparticle subunit. (A)Schematic drawing of E2p and I3-01 nanoparticle design with apan-reactive T-helper epitope fused to the C-terminus of thenanoparticle subunit. (B) SEC profiles of BG505 gp140 trimer-presentingE2p and I3-01 nanoparticles obtained from a Superose 6 10/300 GL columnafter purification using a 2G12 antibody affinity column. (C) Rawmicrographs of BG505 gp140 trimer-presenting E2p and I3-01 nanoparticlesobtained from negative-stain EM.

DETAILED DESCRIPTION I. Overview

The present invention is predicated in part on the present inventors'development of novel HIV-1 gp140 nanoparticle immunogens. As detailed inthe Examples herein, the inventors utilized a T-helper epitope that actsnot only as the linker between gp140 and displaying nanoparticlescaffold, but also as an embedded T-help signal to induce robust T-cellresponses and to steer B cell development towards bNAbs. The inventorsadditionally explored a previously unutilized protein (1VLW) to providestable nanoparticle scaffold in presenting HIV-1 gp140 trimers. Uponpurification with affinity column and size-exclusion chromatography, thevarious scaffolded HIV-1 gp140 immunogens display excellent purity andhomogeneity. When evaluated with bNAbs and non-NAbs, the novel HIV-1gp140 nanoparticles described herein exhibit an outstanding antigenicprofile with a strong PG16 binding that has not been observed with otherknown HIV-1 gp140 nanoparticles. Further as exemplification,immunogenicity of the nanoparticles displaying a BG505 gp140 trimer wasexamined by immunizing mice and assessing HIV-1 neutralizationactivities of IgG isolated from the mice. Neutralization of autologoustier-2 BG505.N332 HIV-1 virus was observed for two HIV-1 gp140nanoparticles disclosed herein (S2G5 and S2G6), as well as control HIV-1immunogens (a scaffolded gp140.681 trimer (S1G5) and a ferritinnanoparticle (S2G1)). Importantly, the novel HIV-1 gp140 nanoparticleimmunogens described herein yielded an IC50 value indicative of rapiddevelopment of tier-2 NAbs after only 8 weeks of immunization,presenting the best HIV-1 vaccine candidate identified thus far, withbalanced T- and B-cell responses.

The invention accordingly provides novel scaffolded HIV-1 vaccineimmunogens harboring a T-helper epitope as exemplified herein. Alsoprovided in the invention are scaffolded HIV-1 vaccine immunogenscontaining stable nanoparticle formed with 1VLW variants. The inventionadditionally provides therapeutic and preventive applications of thesenovel scaffolded HIV-1 immunogens in the treatment or prevention ofHIV-1 infections.

Unless otherwise specified herein, the vaccine immunogens of theinvention, the encoding polynucleotides, expression vectors and hostcells, as well as the related therapeutic applications, can all begenerated or performed in accordance with the procedures exemplifiedherein or routinely practiced methods well known in the art. See, e.g.,Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N.Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1stedition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos. 4,965,343, and5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y. (3^(rd) ed., 2000); Brent et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed.,2003); Davis et al., Basic Methods in Molecular Biology, ElsevierScience Publishing, Inc., New York, USA (1986); or Methods inEnzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Bergerand A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987);Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al.,ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology(CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), andCulture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney,Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods(Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barneseditors, Academic Press, 1st edition, 1998). The following sectionsprovide additional guidance for practicing the compositions and methodsof the present invention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler(Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry,Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and ADictionary of Biology (Oxford Paperback Reference), Martin and Hine(Eds.), Oxford University Press (4th ed., 2000). Further clarificationsof some of these terms as they apply specifically to this invention areprovided herein.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, “an Env-derived trimer” can refer to both singleor plural Env-derived trimer molecules, and can be considered equivalentto the phrase “at least one Env-derived trimer.”

Unless otherwise noted, the terms “antigen” and “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The terms alsorefer to proteins that are immunologically active in the sense that onceadministered to a subject (either directly or by administering to thesubject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein. Thus, in some embodiments, the term“immunogen” can broadly encompass polynucleotides that encodepolypeptide or protein antigens described herein.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given protein. For polypeptide sequences, “conservativelymodified variants” refer to a variant which has conservative amino acidsubstitutions, amino acid residues replaced with other amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Epitope refers to an antigenic determinant. These are particularchemical groups or peptide sequences on a molecule that are antigenic,such that they elicit a specific immune response, for example, anepitope is the region of an antigen to which B and/or T cells respond.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein.

Effective amount of a vaccine or other agent that is sufficient togenerate a desired response, such as reduce or eliminate a sign orsymptom of a condition or disease, such as AIDS. For instance, this canbe the amount necessary to inhibit viral replication or to measurablyalter outward symptoms of the viral infection, such as increase of Tcell counts in the case of an HIV-1 infection. In general, this amountwill be sufficient to measurably inhibit virus (for example, HIV)replication or infectivity. When administered to a subject, a dosagewill generally be used that will achieve target tissue concentrations(for example, in lymphocytes) that has been shown to achieve in vitroinhibition of viral replication. In some examples, an “effective amount”is one that treats (including prophylaxis) one or more symptoms and/orunderlying causes of any of a disorder or disease, for example to treatHIV. In one example, an effective amount is a therapeutically effectiveamount. In one example, an effective amount is an amount that preventsone or more signs or symptoms of a particular disease or condition fromdeveloping, such as one or more signs or symptoms associated with AIDS.

Ferritin is a globular protein found in all animals, bacteria, andplants. It acts primarily to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. The globular formof ferritin is made up of monomeric subunit proteins (also referred toas monomeric ferritin subunits), which are polypeptides having amolecule weight of approximately 17-20 kDa.

As used herein, a fusion protein is a recombinant protein containingamino acid sequence from at least two unrelated proteins that have beenjoined together, via a peptide bond, to make a single protein. Theunrelated amino acid sequences can be joined directly to each other orthey can be joined using a linker sequence. As used herein, proteins areunrelated, if their amino acid sequences are not normally found joinedtogether via a peptide bond in their natural environment(s) (e.g.,inside a cell). For example, the amino acid sequences of monomericsubunits that make up ferritin, and the amino acid sequences of HIV-1gp120 or gp41 glycoproteins are not normally found joined together via apeptide bond.

HIV-1 envelope protein (Env) is initially synthesized as a longerprecursor protein of 845-870 amino acids in size, designated gp160.gp160 forms a homotrimer and undergoes glycosylation within the Golgiapparatus. In vivo, gp160 glycoprotein is endo-proteolytically processedto the mature envelope glycoproteins gp120 and gp41, which arenoncovalently associated with each other in a complex on the surface ofthe virus. The gp120 surface protein contains the high affinity bindingsite for human CD4, the primary receptor for HIV, as well as domainsthat interact with fusion coreceptors, such as the chemokine receptorsCCR5 and CXCR4. The gp41 protein spans the viral membrane and containsat its amino-terminus a sequence of amino acids important for the fusionof viral and cellular membranes. The native, fusion-competent form ofthe HIV-1 envelope glycoprotein complex is a trimeric structure composedof three gp120 and three gp41 subunits. The receptor-binding (CD4 andco-receptor) sites are located in the gp120 moieties, whereas the fusionpeptides are located in the gp41 components. Exemplary sequence ofwildtype gp160 polypeptides are shown in GenBank, e.g., under accessionnumbers AAB05604 and AAD12142.

gp140 refers to an oligomeric form of HIV envelope protein, whichcontains all of gp120 and the entire gp41 ectodomain. As used herein, aHIV-1 gp140 trimer immunogen typically contains a gp140 domain and amodified or redesigned ectodomain of gp140 (gp41_(ECTO)).

gp120 is an envelope protein of the Human Immunodeficiency Virus (HIV).gp120 contains most of the external, surface-exposed, domains of the HIVenvelope glycoprotein complex, and it is gp120 which binds both tocellular CD4 receptors and to cellular chemokine receptors (such asCCR5). The mature gp120 wildtype polypeptides have about 500 amino acidsin the primary sequence. Gp120 is heavily N-glycosylated giving rise toan apparent molecular weight of 120 kD. The polypeptide is comprised offive conserved regions (C1-05) and five regions of high variability(V1-V5). In its tertiary structure, the gp120 glycoprotein is comprisedof three major structural domains (the outer domain, the inner domain,and the bridging sheet) plus the variable loops. See, e.g., Wyatt etal., Nature 393, 705-711, 1998; and Kwong et al., Nature 393, 649-59,1998. The inner domain is believed to interact with the gp41 envelopeglycoprotein, while the outer domain is exposed on the assembledenvelope glycoprotein trimer.

Variable region 1 and Variable Region 2 (V1N2 domain) of gp120 arecomprised of about 50-90 residues which contain two of the most variableportions of HIV-1 (the V1 loop and the V2 loop), and one in ten residuesof the V1N2 domain are N-glycosylated.

gp41 is a proteolytic product of the precursor HIV envelope protein. Itcontains an N-terminal fusion peptide (FP), a transmembrane domain, aswell as an ectodomain that links the fusion peptide and a transmembranedomain. gp41 remains in a trimeric configuration and interacts withgp120 in a non-covalent manner. The amino acid sequence of an exemplarygp41 is set forth in GenBank, under Accession No. CAD20975.

BG505 SOSIP.664 gp140 is a HIV-1 Env immunogen developed with the gp140trimer from clade-A strain BG505. It contains a covalent linkage betweenthe cleaved gp120 and gp41_(ECTO) with an engineered disulfide bond(termed SOS). In addition, it has an I559P mutation (termed IP) todestabilize the gp41 post-fusion conformation and also a truncation ofthe membrane-proximal external region (MPER) at residue 664 to improvesolubility. This HIV-1 immunogen has an outstanding antigenic profileand excellent structural mimicry of the native spike. Using the SOSIPtrimer as a sorting probe, new bNAbs have been identified andcharacterized. The SOSIP design has also been extended to other HIV-1strains and permitted the incorporation of additional stabilizingmutations. Recently, immunogenicity of SOSIP trimers in rabbits andnonhuman primates was reported, paving the way for human vaccine trials.

Immune response refers to a response of a cell of the immune system,such as a B cell, T cell, or monocyte, to a stimulus. In someembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In some embodiments, an immune response isa T cell response, such as a CD4+ response or a CD8+ response. In someother embodiments, the response is a B cell response, and results in theproduction of specific antibodies.

Immunogenic composition refers to a composition comprising animmunogenic polypeptide that induces a measurable CTL response againstvirus expressing the immunogenic polypeptide, or induces a measurable Bcell response (such as production of antibodies) against the immunogenicpolypeptide.

Sequence identity or similarity between two or more nucleic acidsequences, or two or more amino acid sequences, is expressed in terms ofthe identity or similarity between the sequences. Sequence identity canbe measured in terms of percentage identity; the higher the percentage,the more identical the sequences are. Two sequences are “substantiallyidentical” if two sequences have a specified percentage of amino acidresidues or nucleotides that are the same (i.e., 60% identity,optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

Homologs or orthologs of nucleic acid or amino acid sequences possess arelatively high degree of sequence identity/similarity when alignedusing standard methods. Methods of alignment of sequences for comparisonare well known in the art. Various programs and alignment algorithms aredescribed in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman& Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988;Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994.Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailedconsideration of sequence alignment methods and homology calculations.

The term “subject” refers to any animal classified as a mammal, e.g.,human and non-human mammals. Examples of non-human animals include dogs,cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unlessotherwise noted, the terms “patient” or “subject” are used hereininterchangeably. Preferably, the subject is human.

The term “treating” or “alleviating” includes the administration ofcompounds or agents to a subject to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease (e.g., anHIV infection), alleviating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. Subjects inneed of treatment include those already suffering from the disease ordisorder as well as those being at risk of developing the disorder.Treatment may be prophylactic (to prevent or delay the onset of thedisease, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease.

Uncleaved pre-fusion-optimized (UFO) trimers refer to HIV-1 gp140trimeric proteins that are formed with gp120 protein and a redesignedgp41_(ECTO) domain, which results in more stabilized HIV-1 gp140trimers. The redesigned gp41_(ECTO) domain is based on the prototypeHIV-1 strain BG505 (and the prototype gp140 trimer BG505 SOSIP.664gp140) and contains one or more modifications relative to the wildtypeBG505 gp41_(ECTO) sequence. These modifications include (1) replacementof the 21 residue N-terminus of HR1 (residues 548-568) with a shorterloop sequence to stabilize the pre-fusion gp140 structure and (2)replacement of the furin cleavage site between gp120 and gp41 (residues508-511) with a flexible linker sequence such a tandem repeat of a GGGGS(SEQ ID NO:4) motif. In some embodiments, the UFO trimer canadditionally contain an engineered disulfide bond between gp120 and gp41and/or a stabilizing mutation in gp41. For example, UFO trimers based onHIV-1 strain BG505 can contain an engineered disulfide bond is betweenresidues A501C and T605C, and/or a stabilizing mutation I559P. Detaileddescription of UFO trimers is provided in, e.g., Kong et al., Nat. Comm.7:12040, 2016. In addition to UFO trimers based on the BG505 strainsequence, the engineered gp41_(ECTO) domain can be used to pair with agp120 polypeptide from many different HIV-1 strains or subtypes to form“chimeric” gp140 trimers. Such chimeric trimers are termed “UFO-BG” or“UFO²-BG” as exemplified herein.

Vaccine refers to a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective immune response. Typically, avaccine elicits an antigen-specific immune response to an antigen of apathogen, for example a viral pathogen, or to a cellular constituentcorrelated with a pathological condition. A vaccine may include apolynucleotide (such as a nucleic acid encoding a disclosed antigen), apeptide or polypeptide (such as a disclosed antigen), a virus, a cell orone or more cellular constituents.

Virus-like particle (VLP) refers to a non-replicating, viral shell,derived from any of several viruses. VLPs are generally composed of oneor more viral proteins, such as, but not limited to, those proteinsreferred to as capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. Forexample, VLPs can be isolated by density gradient centrifugation and/oridentified by characteristic density banding. Alternatively,cryoelectron microscopy can be performed on vitrified aqueous samples ofthe VLP preparation in question, and images recorded under appropriateexposure conditions.

III. Novel Scaffolded HIV-1 Trimer Immunogens

The invention provides HIV-1 immunogens that contain a heterologousscaffold that presents or incorporates a trimeric HIV-1 Env-derivedprotein (e.g., gp140 trimer) and also a T-helper or linker sequence. Insome embodiments, the heterologous presenting scaffold is aself-assembling nanoparticle. In some other embodiments, theheterologous presenting scaffold is a virus-like particle (VLP) such asbacteriophage Q_(β) VLP. In some embodiments (as exemplified in Example1 herein), subunit of the trimeric HIV-1 protein is linked to theN-terminus of the subunit of the displaying scaffold (e.g.,nanoparticle) via a linker sequence described herein, e.g., a T-helperepitope polypeptide that also functions to promote T cell recognition ofthe fusion immunogen. In some other embodiments (as exemplified inExample 7 herein), subunit of the HIV-1 trimer protein is connected(e.g., covalently linked) to the N-terminus of subunit of the displayingscaffold, and a T-helper or linker epitope is fused to the C-terminus ofsubunit of the displaying scaffold (e.g., nanoparticle). In the latterembodiments, the T-helper epitope can be fused to the nanoparticlesubunit via a short peptide spacer. This allows the formation of ahydrophobic core inside the nanoparticle that functions to stabilize thenanoparticle structure and to promote T cell recognition of the fusionimmunogen. In various embodiments, the short peptide spacer can be,e.g., 1-5 repeats of GGGGS (SEQ ID NO:4), GSGSG (SEQ ID NO:19), or anypeptide that is structurally flexible by nature. As exemplification,T-helper epitope PADRE can be fused to the C-terminus of the subunit ofE2p and I3-01 with a 5-aa GGGGS spacer (Example 7). In addition to usinga short peptide spacer to fuse the T-helper epitope to the C-terminus ofthe nanoparticle subunit, a second peptide spacer or segment can be usedto fuse the HIV-1 trimer to the N-terminus of the nanoparticle subunit.For example, the HIV-1 protein can be fused to the N-terminus of thedisplaying nanoparticle subunit via, e.g., a single glycine residue (“1Glinker”) or 10-aa GGGGSGGGGS (SEQ ID NO:20) spacer exemplified herein(Example 7).

Any Env-derived HIV-1 trimer proteins can be used in thenanoparticle-presented vaccine compositions. The Env-derived trimerprotein can be obtained from various HIV-1 strains. In some embodiments,the nanoparticles present a native trimeric form of HIV-1 Env basedglycoproteins or domains, e.g., gp140, gp120 or V1V2 domains. In someembodiments, the Env-derived trimer is from HIV-1 strain BG505, e.g.,the BG505. SOSIP.664 gp140 trimer. In some embodiments, thenanoparticles present a modified gp140 trimer immunogen, e.g., aHR1-modified gp140 trimer (“UFO trimer”) described in Kong et al., Nat.Comm. 7, 12040, 2016. In some embodiments, the HIV-1 trimeric immunogenused in the invention is a UFO²—BG trimer as exemplified herein. UFO²—BGtrimers are chimeric gp140 trimers containing (1) the BG505 gp41 domainwith a redesigned HR1 N-terminal bend and a cleavage-site linker (asdescribed in Kong et al., Nat. Comm. 7, 12040, 2016) and (2) the gp120protein from one of other diverse HIV-1 strains or subtypes. In additionto the redesigned gp41_(ECTO) domain from the BG505 strain, the gp41domain in the chimeric gp140 trimers suitable for the invention can alsobe a consensus gp41_(ECTO) domain derived from the HIV-1 sequencedatabase.

In various embodiments, nanparticle displaying any of these HIV-1Env-derived immunogens can be constructed by fusing the trimer immunogento the subunit of the nanoparticle (e.g., I3-01, 1VLW derivedpolypeptide sequences or ferritin subunit). The antigeniciy andstructural integrity of these nanoparticle based HIV-1 immunogens can bereadily analyzed via standard assays, e.g., antibody binding assays andnegative-stain electron microscopy (EM). As exemplified herein, thevarious fusion molecules can all self-assemble into nanoparticles thatdisplay immunogenic epitopes of the Env-derived trimer (e.g., gp140). Byeliciting a robust trimer-specific bnAbs, these nanoparticles are usefulfor vaccinating individuals against a broad range of HIV-1 viruses.

In some embodiments, the scaffolded gp140 trimer immunogens of theinvention contain a T-helper epitope that functions as a linker toconnect the gp140 trimer to the nanoparticle scaffold. In some otherembodiments, the T-helper epitope is fused to the C-terminus of thenanoparticle subunit via a short peptide spacer and is encapsulatedinside the nanoparticle scaffold. The short peptide spacer that can beemployed in these embodiments can be, e.g., GGGGS, GSGSG, or any otherpeptide that is structurally flexible by nature. In addition to its roleas a structural element of the scaffolded immunogen, the T-helperepitope also provides an embedded T-help signal to induce robust T-cellresponses and to steer B cell development towards bNAbs. Any T-helperepitope sequences or peptides known in the art may be employed in thepractice of the present invention. They include any polypeptide sequencethat contain MHC class-II epitopes and can effectively activate helper Tcells upon immunization. See, e.g., Alexander et al., Immunity 1,751-761, 1994; Ahlers et al., J. Clin. Invest. 108:1677-1685, 2001;Fraser et al., Vaccine 32, 2896-2903, 2014; De Groot et al., Immunol.Cell Biol. 8:255-269, 2002; and Gene Ther. 21: 225-232, 2014. In somepreferred embodiments, the employed T-helper epitope is a universal panDR epitope peptide (PADRE). In some of these embodiments, the linkercontains a sequence AKFVAAWTLKAAA (SEQ ID NO:1), a conservativelymodified variant or substantially identical (e.g., at least 90%, 95% or99% identical) sequence thereof. In some other embodiments, the employedT-helper epitope is the D T-helper epitope QSIALSSLMVAQAIP (SEQ ID NO:2)or the TpD epitope ILMQYIKANSKFIGIPMGLPQSIALSSLMVAQ (SEQ ID NO:3). Invarious embodiments, the linker can contain a sequence as shown in SEQID NO:2 or SEQ ID NO:3, a substantially identical (e.g., at least 90%,95% or 99% identical) sequence or a conservatively substituted sequencethereof.

As noted above, the heterologous scaffold for presenting or displayingthe trimeric HIV-1 protein is preferably a nanoparticle. Variousnanoparticle platforms can be employed in generating the vaccinecompositions of the invention. In general, the nanoparticles employed inthe invention need to be formed by multiple copies of a single subunit.Additionally or alternatively, the amino-terminus of the particlesubunit has to be exposed and in close proximity to the 3-fold axis, andthe spacing of three amino-termini has to closely match the spacing ofthe carboxyol-termini of various HIV-1 trimeric components. In somepreferred embodiments, the immunogens comprise self-assemblingnanoparticles with a diameter of about 20 nm or less (usually assembledfrom 12, 24, or 60 subunits) and 3-fold axes on the particle surface.Such nanoparticles provide suitable particle platforms to producemultivalent HIV-1 trimer vaccines.

In some embodiments, the scaffolded gp140 trimer immunogens of theinvention are constructed with a hyperstable nanoparticle scaffold. Forexample, the self-assembly nanoparticle can be generated with the I3-01protein described in Hsia et al., Nature 535, 136-139, 2016. The aminoacid sequence of this protein is shown in SEQ ID NO:18. In some otherembodiments, the hyperstable nanoparticle scaffold may be based on avariant of I3-01 as described in Hsia et al. (supra), including aconservatively modified variant or one with a substantially identical(e.g., at least 90%, 95% or 99% identical) sequence. In someembodiments, the linker sequence for connecting the gp140 trimer to theI3-01 derived nanoparticle platform contains a T-helper epitope asdescribed above. In some other embodiments, a glycine-serine polypeptideis used as a second peptide spacer for connecting the gp140 trimer tothe I3-01 derived nanoparticle platform, and a T-helper epitope is fusedto the C-terminus of the nanoparticle subunit via a short peptidespacer. Such a structural design leads to the creation of a hydrophobiccore inside the nanoparticle, which enhances T-cell recognition of thegp140 trimer displayed on the nanoparticle surface. In variousembodiments, the short peptide spacer for linking the T-helper epitopeto the C-terminus of the nanoparticle subunit can be, e.g., GGGGS,GSGSG, or any other peptide that is structurally flexible by nature.

I3-01 sequence (SEQ ID NO: 18):MHHHHHHGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE

In some embodiments, the hyperstable nanoparticles in the scaffoldedgp140 trimer immunogens of the invention are constructed with ferritin,a natural nanoparticle from Helicobacter pylori. For example, thescaffolded gp140 trimer immunogens can contain a UFO²—BG trimer that islinked to and presented on ferritin. In some of these embodiments, theUFO2-BG trimer is directly connected to the ferritin subunit without alinker sequence, as exemplified herein. In some other embodiments, alinker sequnece such as a T-helper epitope or a simple glycine-serinelinker may be used. In some of these embodiments, a T-helper epitope canbe fused to the C-terminus of a nanoparticle subunit via a short peptidespacer, which results in the formation of a hydrophobic core within thenanoaprtilce scaffold. As described herein, the short peptide spacerused in these embodiments can be, e.g., GGGGS, GSGSG, or any otherpeptide that is structurally flexible by nature. Ferritin is a globularprotein found in all animals, bacteria, and plants. The globular form offerritin is made up of monomeric subunit proteins (also referred to asmonomeric ferritin subunits), which are polypeptides having a moleculeweight of approximately 17-20 kDa. A monomeric ferritin subunit used inthe invention is a full length, single polypeptide of a ferritinprotein, or any portion thereof, which is capable of directingself-assembly of monomeric ferritin subunits into the globular form ofthe protein. Amino acid sequences from monomeric ferritin subunits ofany known ferritin protein can be used to produce fusion proteins of thepresent invention, so long as the monomeric ferritin subunit is capableof self-assembling into a nanoparticle displaying HIV-1 epitopes on itssurface. In addition to ferritin, the invention can also employ manyother self-assembling nanoparticles with similar molecular traits. Theseinclude, e.g., molecules with the following PDB IDs: 1JIG (12-mer Dlp-2from Bacillus anthraces), 1UVH (12-mer DPS from MycrobacteriumSmegmatis), 2YGD (24-mer eye lens chaperone aB-crystallin), 3CSO (24-merDegP24), 3MH6 and 3MH7 (24-mer HtrA proteases), 3PV2 (12-mer HtrAhomolog DegQ WT), 4A8C (12-mer DegQ from E. Coli.), 4A9G (24-mer DegQfrom E. Coli.), 4EVE (12-mer HP-NAP from Helicobacter pylori strainYS29), and 4GQU (24-mer HisB from Mycobacterium tuberculosis).

In some embodiments, the scaffolded gp140 trimer immunogens of theinvention can be constructed with a nanoparticle scaffold that isderived from protein 1VLW (SEQ ID NO:5) or its variants as exemplifiedherein (SEQ ID NOs:6-17). In various embodiments, the nanoparticleplatform for constructing the scaffolded gp140 immunogens of theinvention can be produced with a polypeptide sequence that is aconservatively modified variant or a substantially identical sequence ofany one of SEQ ID NOs:5-18. In some embodiments, the linker sequence forconnecting the gp140 trimer to the 1VLW derived nanoparticle platformcontains a T-helper epitope as described above. In some otherembodiments, the linker for connecting the gp140 trimer to the 1VLWderived nanoparticle platform contains a simple peptide sequence. Forexample, the scaffolded immunogens can be constructed with aglycine-serine linker, e.g., a linker that contains 1 to 5 repeats(e.g., 1 or 2 repeats) of GGGGS (SEQ ID NO:4) or GSGSG (SEQ ID NO:19).In some other embodiments, a T-helper epitope can be fused to theC-terminus of the nanoparticle subunit via a short peptide spacer toform a hydrophobic core inside the nanoparticle. In various embodiments,the employed short peptide spacer can be, e.g., GGGGS, GSGSG, or anyother peptide that is structurally flexible by nature.

In some other embodiments, the nanoparticle scaffold for presentingHIV-1 trimer immunogens are redesigned variants of dihydrolipoylacyltransferase (E2p) from Bacillus stearothermophilus. E2p is athermostable 60-meric nanoparticle with a diameter of 23.2 nm and 12large openings separating the threefold vertices on the particlesurface. Nanoparticles formed with the redesigned E2p variants forconstructing the scaffolded HIV-1 trimer immunogens of the inventionhave enhanced stability relative to the wildtype E2p nanoparticles. Insome embodiments, the HIV-1 gp140 trimer can be connected to E2pnanoparticle with a linker that contains a T-helper epitope describedabove. In some other embodiments, a T-helper epitope can be fused to theC-terminus of the E2p subunit via a short peptide spacer so that thefully assembled E2p nanoparticle encapsulate a hydrophobic core formedby the T-helper epitope. The hydrophobic core also functions to enhancethe T-cell recognition of the gp140 trimer on the E2p nanoparticlesurface. The short peptide spacer suitable for use in these embodimentsfor linking E2p and the T-helper epitope can be, e.g., GGGGS, GSGSG, orany other peptide that is structurally flexible by nature.

The scaffolded HIV-1 trimer immunogens of the invention can beconstructed in accordance with the protocols described herein (e.g.,Examples 1-7) and/or other methods that have been described in the art,e.g., He et al., Nat. Comm. 7, 12041, 2016; and Kong et al., Nat. Comm.7, 12040, 2016.

IV. Vectors and Host Cells for Expressing HIV-1 Vaccine Immunogens

The invention provides polynucleotide sequences that encode the HIV-1vaccine immunogens and related polypeptides as described herein,expression vectors that harbor the polynucleotide sequences, as well ashost cells that harbor the polynucleotides or expression constructs. Thecell can be, for example, a eukaryotic cell, or a prokaryotic cell, suchas an animal cell, a plant cell, a bacterium, or a yeast. A variety ofexpression vector/host systems are suitable for expressing the fusionpolypeptides of the invention. Examples include, e.g., microorganismssuch as bacteria transformed with recombinant bacteriophage, plasmid orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors(e.g., baculovirus); plant cell systems transfected with virusexpression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with bacterial expression vectors (e.g., Tior pBR322 plasmid); or animal cell systems.

Vectors useful for the invention preferably contain sequences operablylinked to the fusion polypeptide coding sequences that permit thetranscription and translation of the encoding polynucleotide sequences.Sequences that permit the transcription of the linked fusion polypeptideencoding sequences include a promoter and optionally also include anenhancer element or elements permitting the strong expression of thelinked sequences. The term “transcriptional regulatory sequences” refersto the combination of a promoter and any additional sequences conferringdesired expression characteristics (e.g., high level expression,inducible expression, tissue- or cell-type-specific expression) on anoperably linked nucleic acid sequence. The promoter sequence can beconstitutive or inducible. Examples of constitutive viral promotersinclude the HSV, TK, RSV, SV40 and CMV promoters. Examples of suitableinducible promoters include promoters from genes such as cytochrome P450genes, heat shock protein genes, metallothionein genes,hormone-inducible genes, such as the estrogen gene promoter, and thelike.

In addition to promoter/enhancer elements, expression vectors of theinvention may further comprise a suitable terminator. Such terminatorsinclude, for example, the human growth hormone terminator, or, for yeastor fungal hosts, the TPI1 (Alber & Kawasaki, J Mol Appl Genet. 1:419-34,1982) or ADH3 terminator (McKnight et al., 1985, EMBO J. 4: 2093-2099).Vectors useful for the invention may also comprise polyadenylationsequences (e.g., the SV40 or Ad5E1b poly(A) sequence), and translationalenhancer sequences (e.g., those from Adenovirus VA RNAs). Further, avector useful for the invention may encode a signal sequence directingthe fusion polypeptide to a particular cellular compartment or,alternatively, may encode a signal directing secretion of the fusionpolypeptide.

In some preferred embodiments, vectors expressing the vaccine immunogensof the invention are viral vectors for mammalian expression. In general,any viral vector that permits the introduction and expression ofsequences encoding the fusion HIV-immunogens of the invention isacceptable for the invention. In various embodiments, mammalianexpression vectors can be used in the practice of the invention,including the adenoviral vectors, the pSV and the pCMV series of plasmidvectors, vaccinia and retroviral vectors, as well as baculovirus. Forexample, the HIV-1 vaccine immunogens of the invention can be expressedfrom viral vector phCMV3.

Depending on the specific vector used for expressing the fusionpolypeptide, various known cells or cell lines can be employed in thepractice of the invention. The host cell can be any cell into whichrecombinant vectors carrying a fusion HIV-immunogen of the invention maybe introduced and wherein the vectors are permitted to drive theexpression of the fusion polypeptide is useful for the invention. It maybe prokaryotic, such as any of a number of bacterial strains, or may beeukaryotic, such as yeast or other fungal cells, insect or amphibiancells, or mammalian cells including, for example, rodent, simian orhuman cells. Cells expressing the fusion polypeptides of the inventionmay be primary cultured cells, for example, primary human fibroblasts orkeratinocytes, or may be an established cell line, such as NIH3T3,HEK293, HEK293T HeLa, MDCK, WI38, or CHO cells. In some embodiments, thehost cells for expressing the HIV-1 vaccine immunogens of the inventioncan be ExpiCHO cells or HEK293F cells as exemplified herein. The skilledartisans can readily establish and maintain a chosen host cell type inculture that expresses the fusion vaccine immunogens of the invention.Many other specific examples of suitable cell lines that can be used inexpressing the fusion polypeptides are described in the art. See, e.g.,Smith et al., 1983., J. Virol 46:584; Engelhard, et al., 1994, Proc NatAcad Sci 91:3224; Logan and Shenk, 1984, Proc Natl Acad Sci, 81:3655;Scharf, et al., 1994, Results Probl Cell Differ, 20:125; Bittner et al.,1987, Methods in Enzymol, 153:516; Van Heeke & Schuster, 1989, J BiolChem 264:5503; Grant et al., 1987, Methods in Enzymology 153:516;Brisson et al., 1984, Nature 310:511; Takamatsu et al., 1987, EMBO J6:307; Coruzzi et al., 1984, EMBO J 3:1671; Broglie et al., 1984,Science, 224:838; Winter J and Sinibaldi R M, 1991, Results Probl CellDiffer., 17:85; Hobbs S or Murry L E in McGraw Hill Yearbook of Scienceand Technology (1992) McGraw Hill New York N.Y., pp 191-196 or Weissbachand Weissbach (1988) Methods for Plant Molecular Biology, AcademicPress, New York, pp 421-463.

The fusion polypeptide-expressing vectors may be introduced to selectedhost cells by any of a number of suitable methods known to those skilledin the art. For the introduction of fusion polypeptide-encoding vectorsto mammalian cells, the method used will depend upon the form of thevector. For plasmid vectors, DNA encoding the fusion polypeptidesequences may be introduced by any of a number of transfection methods,including, for example, lipid-mediated transfection (“lipofection”),DEAE-dextran-mediated transfection, electroporation or calcium phosphateprecipitation. These methods are detailed, for example, in Brent et al.,supra. Lipofection reagents and methods suitable for transienttransfection of a wide variety of transformed and non-transformed orprimary cells are widely available, making lipofection an attractivemethod of introducing constructs to eukaryotic, and particularlymammalian cells in culture. For example, LipofectAMINE™ (LifeTechnologies) or LipoTaxi™ (Stratagene) kits are available. Othercompanies offering reagents and methods for lipofection include Bio-RadLaboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBIFermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, andWako Chemicals USA.

For long-term, high-yield production of recombinant fusion polypeptides,stable expression is preferred. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with the fusion polypeptide-encoding sequences controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), andselectable markers. The selectable marker in the recombinant vectorconfers resistance to the selection and allows cells to stably integratethe vector into their chromosomes. Commonly used selectable markersinclude neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene, 30: 147,1984). Through appropriate selections, the transfected cells can containintegrated copies of the fusion polypeptide encoding sequence.

V. Pharmaceutical Compositions and Therapeutic Applications

The invention provides pharmaceutical compositions and related methodsof using the scaffolded HIV-1 immunogen polypeptides or polynucleotidesencoding the vaccine polypeptides described herein for preventing andtreating HIV-1 infections. In some embodiments, the immunogens disclosedherein are included in a pharmaceutical composition. The pharmaceuticalcomposition can be either a therapeutic formulation or a prophylacticformulation. Typically, the composition additionally includes one ormore pharmaceutically acceptable vehicles and, optionally, othertherapeutic ingredients (for example, antibiotics or antiviral drugs).Various pharmaceutically acceptable additives can also be used in thecompositions.

Some of the pharmaceutical compositions of the invention are vaccines.For vaccine compositions, appropriate adjuvants can be additionallyincluded. Examples of suitable adjuvants include, e.g., aluminumhydroxide, lecithin, Freund's adjuvant, MPL™ and IL-12. In someembodiments, the scaffolded HIV-1 immunogens disclosed herein can beformulated as a controlled-release or time-release formulation. This canbe achieved in a composition that contains a slow release polymer or viaa microencapsulated delivery system or bioadhesive gel. The variouspharmaceutical compositions can be prepared in accordance with standardprocedures well known in the art. See, e.g., Remington's PharmaceuticalSciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa., 1995;Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and4,917,893; 4,677,191 and 4,728,721; and 4,675,189.

Pharmaceutical compositions of the invention can be readily employed ina variety of therapeutic or prophylactic applications for treating HIV-1infection or eliciting an immune response to HIV-1 in a subject. Forexample, the composition can be administered to a subject to induce animmune response to HIV-1, e.g., to induce production of broadlyneutralizing antibodies to HIV-1. For subjects at risk of developing anHIV infection, a vaccine composition of the invention can beadministered to provide prophylactic protection against viral infection.Depending on the specific subject and conditions, pharmaceuticalcompositions of the invention can be administered to subjects by avariety of administration modes known to the person of ordinary skill inthe art, for example, intramuscular, subcutaneous, intravenous,intra-arterial, intra-articular, intraperitoneal, or parenteral routes.In general, the pharmaceutical composition is administered to a subjectin need of such treatment for a time and under conditions sufficient toprevent, inhibit, and/or ameliorate a selected disease or condition orone or more symptom(s) thereof. The immunogenic composition isadministered in an amount sufficient to induce an immune responseagainst HIV-1. For therapeutic applications, the compositions shouldcontain a therapeutically effective amount of the scaffolded HIV-1immunogen described herein. For prophylactic applications, thecompositions should contain a prophylactically effective amount of thescaffolded HIV-1 immunogen described herein. The appropriate amount ofthe immunogen can be determined based on the specific disease orcondition to be treated or prevented, severity, age of the subject, andother personal attributes of the specific subject (e.g., the generalstate of the subject's health and the robustness of the subject's immunesystem). Determination of effective dosages is additionally guided withanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.

For prophylactic applications, the immunogenic composition is providedin advance of any symptom, for example in advance of infection. Theprophylactic administration of the immunogenic compositions serves toprevent or ameliorate any subsequent infection. Thus, in someembodiments, a subject to be treated is one who has, or is at risk fordeveloping, an HIV infection, for example because of exposure or thepossibility of exposure to HIV. Following administration of atherapeutically effective amount of the disclosed therapeuticcompositions, the subject can be monitored for HIV-1 infection, symptomsassociated with HIV-1 infection, or both.

For therapeutic applications, the immunogenic composition is provided ator after the onset of a symptom of disease or infection, for exampleafter development of a symptom of HIV-1 infection, or after diagnosis ofHIV-1 infection. The immunogenic composition can thus be provided priorto the anticipated exposure to HIV virus so as to attenuate theanticipated severity, duration or extent of an infection and/orassociated disease symptoms, after exposure or suspected exposure to thevirus, or after the actual initiation of an infection.

The pharmaceutical composition of the invention can be combined withother agents known in the art for treating or preventing HIV infections.These include, e.g., antibodies or other antiviral agents such asnucleoside reverse transcriptase inhibitors, such as abacavir, AZT,didanosine, emtricitabine, lamivudine, stavudine, tenofovir,zalcitabine, zidovudine, and the like, non-nucleoside reversetranscriptase inhibitors, such as delavirdine, efavirenz, nevirapine,protease inhibitors such as amprenavir, atazanavir, indinavir,lopinavir, nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir,and the like, and fusion protein inhibitors such as enfuvirtide and thelike. Administration of the pharmaceutical composition and the knownanti-HIV agents can be either concurrently or sequentially.

The HIV-1 vaccine immunogens or pharmaceutical compositions of theinvention can be provided as components of a kit. Optionally, such a kitincludes additional components including packaging, instructions andvarious other reagents, such as buffers, substrates, antibodies orligands, such as control antibodies or ligands, and detection reagents.An optional instruction sheet can be additionally provided in the kits.

Examples

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Design and Characterization of UFO²-BG Trimers

A major obstacle faced by current trimer designs is the deterioration ofyield, purity, and stability once they are extended from BG505 to otherstrains. The solutions proposed thus far include (1) purificationmethods aimed to separate native-like trimers from misfolded Envproteins, such as bNAb affinity columns, negative selection, multi-cycleSEC, and a combined chromatographic approach; and (2) auxiliarymutations informed by atomic structures or derived from libraryscreening. However, these solutions are empirical by nature and oftenresult in suboptimal outcomes such as reduced trimer yield andunexpected change in Env properties. We previously identified an HR1bend (residues 547-569) as the primary cause of Env metastability (Konget al., Nat. Comm. 7, 12040, 2016). Rational redesign of thisstructurally strained region in gp41_(ECTO) significantly improvedtrimer yield and purity for multiple HIV-1 strains, yet still producedvarying amounts of misfolded Env, suggesting that other regions besidesHR1 also contribute to Env metastability. Thus, uncovering the source ofthese ‘secondary factors of metastability’ may prove crucial to trimerdesign.

We hypothesized that all factors of Env metastability are encoded withingp41_(ECTO), and that BG505 gp41_(ECTO) of the UFO design (termedUFO²-BG) can be used to stabilize diverse HIV-1 Envs. To examine thishypothesis, we selected ten Envs of five clade origins (A, B, C, B/C,and A/E) from either a large panel of HIV-1 pseudoviruses or theavailable Los Alamos National Laboratory (LANL) database, and alsoincluded three Envs tested in our previous study (Kong et al., Nat.Comm. 7, 12040, 2016). Of note, seven of the ten Envs tested here werederived from tier-2/3 isolates, posing a significant challenge to trimerstabilization. For each Env, the gp140 constructs of SOSIP, UFO, andUFO²-BG designs were expressed transiently in ExpiCHO cells, with furinco-transfected for the SOSIP construct. Following GNL purification, theSEC profiles of thirty gp140s were generated from a Superdex 200 16/600column for comparison. With the exception of BG505, all SOSIPs showed asignificant proportion of aggregates (40-50 ml), which was accompaniedby extremely low yield and sometimes absence of the trimer peak. UFOsnotably improved the trimer yield and purity except for clade A/E, withthe most visible improvement observed for clade C. UFO²-BG demonstratedoutstanding trimer purity and yield for eight of ten strains with no orslight hints of dimers and monomers, covering all seven tier-2/3isolates. All thirty gp140 constructs were then characterized byBN-PAGE. Overall, UFO²-BG dramatically reduced the dimer and monomercontents with respect to SOSIP and UFO, showing a trimer band across SECfractions, but occasionally with a faint band of lower molecular weight.Based on this finding, compared the total Env protein obtained from aGNL column against the trimer portion after subsequent SEC and fractionanalysis by BN-PAGE. Surprisingly, a simple step of GNL purificationyielded comparable purity for all but two UFO²-BG trimers derived from atier-2 clade-B strain and a tier-3 clade-B/C strain. Next, thermalstability was assessed for eight purified UFO²-BG trimers bydifferential scanning calorimetry (DSC). Notably, the DSC profilesexhibited a clade/strain-specific pattern, with the thermal denaturationmidpoint (T_(m)) ranging from 60.9 to 68.4° C. Among the eight trimerstested, BG505—for which UFO²-BG is equivalent to UFO—yielded the highestT_(m) (68.4° C.), which was followed by two clade-C trimers (65.2-66.2°C.). In the absence of additional cavity-filling mutations and disulfidebonds, the DSC data reflected, in large part, the thermal stability ofWT Envs. Of note, the CN54 UFO and UFO²-BG constructs tested herecontains 14 mutations (CN54M14), which reduce aggregates for 293F-produced trimers. In addition, four UFO²-BG trimers of clades B, C,and B/C were selected for expression in 293 F cells and SECpurification. The results indicate that UFO²-BG can improve trimerproperties irrespective of the cell lines used but only reach thehighest purity when used in conjunction with the ExpiCHO system,consistent with our finding for BG505.

The results thus confirm our hypothesis that gp41_(ECTO) is the solesource of Env metastability and BG505 gp41_(ECTO) of the UFO design canstabilize diverse HIV-1 Envs. The nearly identical trimer purity priorto and following the SEC purification suggests a simple, robust, andcost-effective manufacturing process for the UFO²-BG trimers. Theinherent trimer purity will also accelerate the development and clinicaltesting of nucleic acid vaccines expressing the UFO²-BG trimers.

Example 2 Nanoparticle Presentation of UFO²-BG Trimers from DiverseSubtypes

Following our previously reported design strategy (He et al., Nat. Comm.7, 12041, 2016), we investigated whether the UFO²-BG trimers derivedfrom diverse HIV-1 strains can be displayed on the 24-meric ferritin(FR) nanoparticle. We hypothesize that BG505 gp41_(ECTO) of the UFOdesign can facilitate both gp140 trimerization and nanoparticle assembly(FIG. 1A). To test this hypothesis, we designed eight UFO²-BG-FRconstructs with the C terminus of gp41_(ECTO) (residue 664) fused to theN terminus (Asps) of a ferritin subunit. The resulting fusion constructswere expressed transiently in ExpiCHO cells followed by a simplepurification using the 2G12 affinity column. BN-PAGE displayed adistinctive band of high molecular weight corresponding to well-formedUFO²-BG-FR nanoparticles for all eight strains studied. Consistently,nanoparticle assembly was confirmed by negative-stain EM, showing avisible particle core decorated with a regular array of gp140 trimersprotruding from the surface. The DSC analysis indicated high thermalstability for UFO²-BG-FR nanoparticles derived from all five subtypes,with T_(m) ranging from 68 to 70° C. The antigenicity of UFO²-BG-FRnanoparticles was assessed for five representative designs using a panelof six bNAbs and four non-NAbs. Overall, multivalent display hasretained, and in some cases enhanced, the native-like trimerantigenicity, showing patterns specific to the epitopes as well assubtypes. For the V2 apex, all five nanoparticles bound to PGDM1400 withcomparable or notably higher affinity than individual trimers,confirming that trimers displayed on nanoparticle surface adoptnative-like, closed conformations. For H078.14, the restored bNAbbinding could be explained by a shift of conformational equilibriuminfluenced by neighboring trimers on the nanoparticle surface, whereasfor Du172.17 and 93JP NH1 the increased affinity was likely a result ofavidity effect. By contrast, little improvement was observed fornanoparticle binding to another apex bNAb, PG16. For the N332 supersiteand CD4bs, multivalent display exhibited a more favorable effect on theH078.14 UFO²-BG trimer. For the gp120-gp41 interface, while allUFO²-BG-FR nanoparticles retained their trimer binding to PGT151, whichrecruits elements of two adjacent gp140 protomers, a cross-cladereduction of binding signal was observed for 35022, which interacts withonly one protomer. For non-NAbs, nanoparticles exhibited bindingprofiles resembling those of trimers.

We next examined the utility of a 60-unit hyperstable nanoparticle,I3-01 (Hsia et al., Nature 535, 136-139, 2016), for multivalent displayof native-like Env trimers. In terms of symmetry (dodecahedron) and size(25 nm), I3-01 closely resembles the 60-meric E2p nanoparticle tested inour previous study, but with greater stability (FIG. 1E, left). However,the large spacing between the N termini of I3-01 subunits, ˜50.5 Å,requires a long linker to connect with the C termini of the gp140 trimer(FIG. 1E, middle). We hypothesize that a T-helper epitope may be usednot only as a linker between gp140 and an I3-01 subunit but also as anembedded T-help signal to induce robust T-cell responses and to steer Bcell development towards bNAbs. To test this hypothesis, a Pan DRepitope peptide (PADRE), AKFVAAWTLKAAA (SEQ ID NO:1) (Alexander et al.,Immunity 1, 751-761,1994) and two recently reported T-helper epitopes, Dand TpD (Fraser et al., Vaccine 32, 2896-2903, 2014), were selected forevaluation (FIG. 1E, right). Three fusion constructs were designed thatcontain the HR1-redesigned BG505 gp140 (Kong et al., Nat. Comm. 7,12040, 2016), a T-helper epitope, and the I3-01 subunit. Following furinco-expression in ExpiCHO cells, the 2G12-purified material wascharacterized by SEC (FIG. 1F). Remarkably, the I3-01 construct thatcontains PADRE produced high-purity nanoparticles, as further confirmedby BN-PAGE (FIG. 1G) and negative-stain EM (FIG. 1h ). When evaluatedusing the same panel of bNAbs and non-NAbs, this nanoparticle exhibitedan outstanding antigenic profile with a strong PG16 binding that has notbeen observed for any nanoparticles tested thus far (Figure II).

In summary, our results demonstrate that the UFO²-BG trimers of diverseHIV-1 strains can be displayed on ferritin nanoparticle. In addition,the use of a hyperstable nanoparticle such as I3-01 and a T-helperepitope provide a novel platform for developing multivalent HIV-1vaccines with more balanced T and B cell responses.

Example 3 Nanoparticles Potently Activate B Cells Expressing bNAbs

Previously, we demonstrated that various BG505 gp120 and gp140nanoparticles could engage B cells expressing cognate VRC01 receptors(He et al., 2016). In this study, we assessed the B cell activation byfive UFO²-BG-FR nanoparticles and a BG505 gp140-PADRE-I3-01 nanoparticlewith respect to individual trimers (FIG. 2). B cells expressing bNAbsPGT145, VRC01, and PGT121 (Ota et al., J. Immunol. 189, 4816-4824, 2012)were used in the assay. Overall, trimer-presenting nanoparticles couldstimulate bNAb-expressing B cells more effectively than individualtrimers, with peak signals approaching the maximal activation byionomycin. However, the results also revealed a pattern pertinent to theepitope examined: when tested in B cells expressing PGT121, whichrecognize the N332 supersite, some trimers and all nanoparticlesrendered detectable Ca²⁺ flux signals; by contrast, none and few trimersactivated B cells expressing PGT145 and VRC01, which target the V2 apexand the CD4bs, respectively. Of note, the stimulation ofPGT145-expressing B cells by the H078.14 UFO²-BG-FR nanoparticleprovides further evidence that the apex can be restored by multivalentdisplay without V2 mutation, consistent with the BLI data (FIG. 1D). Asimilar effect was also observed for the clade-A/E 93JP NH1 UFO²-BG-FRnanoparticle, which bound to PGT121 only weakly by BLI but induced avisible, long-lasting Ca²⁺ flux signal in PGT121-expressing B cells,suggesting that cross-linking of B cell receptors (BCRs) on cell surfacemay offer additional help to overcome the inherent low affinity.Together, by combining biochemical, structural, and antigenic approacheswith B cell activation assays, we established a panel of gp140nanoparticles that should permit investigation of their vaccinepotential in vivo.

Example 4 Induction of Autologous Neutralizing Antibodies in WildtypeMice

Immunogenicity has been assessed for various forms of native-like Envtrimers. When wild-type mice were immunized with SOSIP trimers, noautologous tier-2 NAb response to BG505.N332 was observed over a periodof eighteen weeks (Hu et al., J. Virol. 89, 10383-10398, 2015). It wasconcluded that the glycan shield of well-formed Env trimers isimpenetrable for murine antibodies due to their short heavy-chaincomplementarity-determining region 3 (HCDR3) loops. Nonetheless, tier-2NAbs were reported to be elicited by modified BG505 SOSIP trimers inmice with knock-in bNAb precursors. Using vaccination regimens spanningsix months to one year, native-like trimers were also reported to inducean autologous tier-2 NAb response in rabbits and a weaker such responsein macaques (de Taeye et al., 2015; Klasse et al., 2016;Martinez-Murillo et al., 2017; Pauthner et al., 2017; Sanders et al.,2015). Therefore, the induction of tier-2 NAbs remains a significantchallenge to HIV-1 vaccine development, particularly in the WT mousemodel.

Here, we immunized the WT BALB/c mice with BG505 gp140 trimers andnanoparticles containing an HR1 redesign—the core of UFO design (Kong etal., 2016a)—using a simple six-week regimen and a serum IgG purificationprocedure to eliminate non-specific antiviral activity (FIG. 3A). PIKA,an human adjuvant that has shown enhanced T-cell and antibody responsesin a phase-I rabies vaccine trial (Wijaya et al., 2017), was used toprovide a human-compatible vaccine formulation. A total of eight trimersand four nanoparticles were tested (FIG. 3B, top), with group-combinedserum IgGs assessed for antigen binding by ELISA (FIG. 3B, bottom). OneV1V2 and two N332 nanoparticle probes were utilized to gauge B cellresponses to the apex and the N332 supersite, respectively (Morris etal., 2017). We first examined mouse IgGs elicited by 293 F andExpiCHO-produced trimers (S1G3 and S1G4), which exhibited differentialbinding to the 293 F-produced probes, confirming the cell line-specificpatterns of glycosylation and B cell response (FIGS. 1D and 1E).Consistent with our previous report (Morris et al., 2017), threescaffolded gp140.681 trimers elicited strong IgG responses in mice, asindicated by the lower EC50 values (S1G5, S1G6, and S1G7). The ferritinnanoparticle (S2G1) appeared to elicit a stronger antibody response tothe N332 supersite, suggesting a positive effect of multivalent display.All three gp140-T-epitope-I3-01 nanoparticles (S2G5, S2G6, and S2G7)outperformed their respective trimers containing PADRE, D, and TpDepitopes at the C terminus (S1G8, S1G9, and S1G10). Lastly, serum IgGsfrom twelve immunized groups were tested for HIV-1 neutralization at anIgG concentration of 3-8 mg/ml in the initial screening, with a naïvegroup included as control (S1G10) (FIG. 3C). In contrast with theprevious negative report (Hu et al., supra), neutralization ofautologous tier-2 BG505.N332 was observed for a scaffolded gp140.681trimer (S1G5), a ferritin nanoparticle (S2G1), and two I3-01nanoparticles (S2G5 and S2G6). When tested at a lower IgG concentration(1 mg/ml), S1G5 showed a borderline neutralization just below thethreshold (FIG. 3D), whereas one subject in S2G1 (FIG. 3E) and twosubjects in S2G5 appeared to have developed NAbs to the autologoustier-2 BG505.N332 (FIG. 3F). In particular, the gp140-PADRE-I3-01nanoparticle not only exhibited outstanding purity, structuralhomogeneity, and antigenicity (FIG. 1, E-I), but also yielded an IC50value indicative of rapid development of tier-2 NAbs after merely eightweeks. These data suggest that immunization with the gp140-PADRE-I3-01nanoparticle could induce a more potent tier-2 NAb response than currenttrimer vaccines, likely also with improved breadth, in rabbits, NHPs,and humans.

Example 5 Other Hyperstable Nanoparticles for Presenting HIV-1 gp140Trimer

In addition to the I3-01 nanoparticle, we also examined other stablenanoparticles for constructing the gp140-T helper epitope-nanoparticleplatform HIV-1 vaccine immunogens described herein. Specifically, wetested a protein “2-Dehydro-3-DeoxyphosphogluconateAldolase4-Hydroxy-2-Oxoglutarate Aldolase (Tm0066) From ThermotogaMaritima” with a 2.30 Å-resolution crystal structure (PDB ID: 1VLW). The1VLW-encoding gene sequence was used as a basis, with the 2.30Å-resolution crystal structure used as the backbone, to design proteinsthat may automatically assemble into 60-meric nanoparticles with moredesirable properties than I3-01. Eleven amino acids within the 1VLWsequence (SEQ ID NO:5) were subjected to the ensemble-based proteindesign, or visual inspection followed by manual design. Twelve designed1VLW mutants were synthesized (SEQ ID NOs:6-17). Construction of gp140trimer displayed on nanoparticles of these sequences, expression of thenanoparticle immunogens, and their immunogenicity are examined via thesame protocols as that described above for the gp140-PADRE-I3-01nanoparticle immunogen.

1VLW wildtype amino acid sequence (SEQ ID NO:5) (residues subject toredesign are underscored):

MKMEELFKKHKIVAVLRANSVEEAK E KA LA VF EG GVHLTETTFTVP DADTVIKELSFLKE KGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVG SALVKGT PD EV RE KA KAFVEKIRGCTE

Redesigned 1VLW variants for displaying gp140 trimer (SEQ ID NOs:6-17)(modified residues are double underlined):

>1VLW-SS1 (SEQ ID NO: 6) MKMEELFKKHKIVAVLRANSVEEAKKKA LA VFL GGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLAVGVGSALVKGTPCEVACKA K AFVEKIRGCTE >1VLW-MUT (SEQ ID NO: 7)MKMEELFKKHKIVAVLRANSVEEAKWKA LA VF I G GVHLIEITFTVPDADTVIKELSFL KE LGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGT PA EV V E KA K AFVEKIRGCTE >1VLW-JZ1 (SEQ ID NO: 8)MKMEELFKKHKIVAVLRANSVEEAK M KA LH VF SG GVHLIEITFTVPDADTVIKELSFL KE QGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTWD EV SR KA K AFVEKIRGCTE >1VLW-JZ2 (SEQ ID NO: 9)MKMEELFKKHKIVAVLRANSVEEAK W KA L H VF T G GVHLIEITFTVPDADTVIKELSFL KE QGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTWH EV AA KA K AFVEKIRGCTE >1VLW-JZ3 (SEQ ID NO: 10)MKMEELFKKHKIVAVLRANSVEEAK M KA L H VF T G GVHLIEITFTVPDADTVIKELSFL KE WGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGT WD EV AA KA K AFVEKIRGCTE >1VLW-JZ4 (SEQ ID NO: 11)MKMEELFKKHKIVAVLRANSVEEAK K KA LA VF LA GVHLIEITFTVPDADTVIKELSFL KE MGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTVV EV AA KA A AFVEKIRGCTE >1VLW-JZ5 (SEQ ID NO: 12)MKMEELFKKHKIVAVLRANSVEEAK K KA LA VF L G GVHLIEITFTVPDADTVIKELSFL KE MGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTIV EV AA KA A AFVEKIRGCTE >1VLW-JZ6 (SEQ ID NO: 13)MKMEELFKKHKIVAVLRANSVEEAK K KA LA VF L G GVHLIEITFTVPDADTVIKELSFL KE MGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTWV EV AA KA A AFVEKIRGCTE >1VLW-JZ7 (SEQ ID NO: 14)MKMEELFKKHKIVAVLRANSVEEAK M KA L Q VF V G GVHLIEITFTVPDADTVIKELSFL KE AGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTLA EV AA KA E AFVEKIRGCTE >1VLW-JZ8 (SEQ ID NO: 15)MKMEELFKKHKIVAVLRANSVEEAK W KA L H VF V G GVHLIEITFTVPDADTVIKELSFL KE AGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTWA EV AA KA K AFVEKIRGCTE >1VLW-JZ9 (SEQ ID NO: 16)MKMEELFKKHKIVAVLRANSVEEAK M KA LA VF V G GVHLIEITFTVPDADTVIKELSFL KE LGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTIA EV AA KA A AFVEKIRGCTE >1VLW-JZ10 (SEQ ID NO: 17)MKMEELFKKHKIVAVLRANSVEEAK M KA LA VF Y G GVHLIEITFTVPDADTVIKELSFL KE AGAIIGAGTVISVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPIGGVNLDNVCEWFKAGVLA VGVGSALVKGTFV EV AA KA A AFVEKIRGCTE

Example 6 Some Exemplified Experimental Procedures

Antibodies: We utilized a panel of bNAbs and non-NAbs to characterizethe antigenicity of various native-like trimers and gp140 nanoparticles.Antibodies were requested from the NIH AIDS Reagent Program except forbNAbs PGDM1400, PGT145, PGT121 and PGT151, and non-NAb 19b, which wereobtained internally in the Scripps Research Institute.

Expression and purification of HIV-1 Env trimers and nanoparticles:Trimers were transiently expressed in HEK293 F or ExpiCHO cells (ThermoFisher) except for materials used for crystallographic analysis. Theprotocol used for trimer production in HEK293 F cells has been describedpreviously (Kong et al., supra; Morris et al., mBio 8, e00036-00017,2017). For cleaved HR1-redesigned trimers, the furin plasmid was addedduring transfection. The protocol used for trimer and nanoparticleproduction in ExpiCHO cells is described as follows. Briefly, ExpiCHOcells were thawed and incubated with ExpiCHO™ Expression Medium (ThermoFisher) in a shaker incubator at 37° C., with 135 rpm and 8% CO₂. Whenthe cells reached a density of 10×10⁶ ml⁻¹, ExpiCHO™ Expression Mediumwas added to reduce cell density to 6×10⁶ ml⁻¹ for transfection. TheExpiFectamine™ CHO/plasmid DNA complexes were prepared for 200 mltransfection in ExpiCHO cells following the manufacturer's instruction.For SOSIP and HR1-redesigned trimers as well as the I3-01 nanoparticlespresenting a BG505 HR1-redesigned trimer, 160 μg of antigen plasmid, 60μg of furin plasmid, and 640 μl of ExpiFectamine™ CHO reagent were mixedin 15.4 ml of cold OptiPRO™ medium (Thermo Fisher), whereas for UFO andUFO² trimers as well as UFO²-BG-FR nanoparticles, 200 μg of antigenplasmid was used without furin. After the first feed on day 1, ExpiCHOcells were cultured in a shaker incubator at 32° C., with 120 rpm and 8%CO₂ following the Max Titer protocol with an additional feed on day 5(Thermo Fisher). Culture supernatants were harvested 13 to 14 days aftertransfection, clarified by centrifugation at 4000 rpm for 20 min, andfiltered using a 0.45 μm filter (Thermo Fisher). For timers, Env proteinwas extracted from the supernatants using a Galanthus nivalis lectin(GNL) column (Vector Labs), whereas for nanoparticles, Env-fusionprotein was purified using a 2G12 affinity column. Trimers might befurther purified by size exclusion chromatography (SEC) on a Superdex200 Increase 10/300 GL column or a HiLoad 16/600 Superdex 200 PG column(GE Healthcare). The purity of I3-03 nanoparticles was characterized bySEC on a Superose 6 10/300 GL column. For both trimers andnanoparticles, protein concentration was determined using UV₂₈₀absorbance with theoretical extinction coefficients.

Analysis of total and site-specific glycosylation profiles: The totalglycan profiles of ExpiCHO and 293 F-produced trimers were generated byHILIC-UPLC. N-linked glycans were enzymatically released from envelopeglycoproteins via in-gel digestion with Peptide-N-Glycosidase F (PNGaseF), subsequently fluorescently labelled with 2-aminobenzoic acid (2-AA)and analyzed by HILIC-UPLC. Digestion of released glycans with Endo Henabled the quantitation of oligomannose-type glycans. The compositionsof the glycans were determined by analyzing released glycans fromtrimers by PNGase F digestion using ion mobility MS. Negative ion mass,collision-induced dissociation (CID) and ion mobility spectra wererecorded with a Waters Synapt G2Si mass spectrometer (Waters Corp.)fitted with a nano-electrospray ion source. Waters Driftscope (version2.8) software and MassLynx™ (version 4.1) was used for data acquisitionand processing. Spectra were interpreted as described previously (Harveyet al., Anal. Biochem. 376, 44-60, 2008). The results obtained served asthe basis for the creation of sample-specific glycan libraries, whichwere used for subsequent site-specific N-glycosylation analyses. Forsite-specific N-glycosylation analysis, before digestion, trimers weredenatured and alkylated by incubation for 1 h at room temperature (RT)in a 50 mM Tris/HCl, pH 8.0 buffer containing 6 M urea and 5 mMdithiothreitol (DTT), followed by the addition of 20 mM iodacetamide(IAA) for a further 1 h at RT in the dark, and then additional DTT (20mM) for another 1 h, to eliminate any residual IAA. The alkylatedtrimers were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspincolumns and digested separately with trypsin and chymotrypsin (MassSpectrometry Grade, Promega) at a ratio of 1:30 (w/w). Glycopeptideswere selected from the protease-digested samples using the ProteoExtractGlycopeptide Enrichment Kit (Merck Millipore). Enriched glycopeptideswere analyzed by LC-ESI MS on an Orbitrap fusion mass spectrometer(Thermo Fisher Scientific), using higher energy collisional dissociation(HCD) fragmentation. Data analysis and glycopeptide identification wereperformed using Byonic™ (Version 2.7) and Byologic™ software (Version2.3; Protein Metrics Inc.).

BN-PAGE: Env proteins and nanoparticles were analyzed by blue nativepolyacrylamide gel electrophoresis (BN-PAGE) and stained with Coomassieblue. The protein samples were mixed with G250 loading dye and added toa 4-12% Bis-Tris NuPAGE gel (Life Technologies). BN-PAGE gels were runfor 2.5 hours at 150 V using the NativePAGE™ running buffer (LifeTechnologies) according to the manufacturer's instructions.

Differential scanning calorimetry (DSC): Thermal stability of UFO²-BGtrimers, UFO²—U trimers, and trimer-presenting nanoparticles wasmeasured using a MicroCal VP-Capillary calorimeter (Malvern) in PBSbuffer at a scanning rate of 90° C. h⁻¹ from 20° C. to 120° C. Data wereanalyzed using the VP-Capillary DSC automated data analysis software.

Protein production and purification for crystallization: The clade-Btier-3 H078.14 UFO²-BG trimer was expressed in FreeStyle 293 S cells andpurified from culture supernatant using a 2G12-coupled affinity matrixfollowed by size exclusion chromatography (SEC). Fabs PGT124 and 35022were transiently transfected into mammalian FreeStyle 293F cells(Invitrogen) and purified using a LC-X capture select column, prior tofurther purification by ion exchange chromatography and SEC on aSuperdex 200 16/60 column. The trimer complexes were prepared by mixingH078.14 UFO²-BG trimer protein with PGT124 and 35022 at a molar ratio of1:3:2 for 30 min at room temperature. To decrease heterogeneity ontrimer complexes, deglycosylation was carried out on H078.14 UFO²-BG.664produced in 293S cells with Endoglycosidase H (New England Biolabs)overnight at 4° C. The trimer complexes were subjected to crystal trialsafter further purification of the complexes by SEC.

Protein crystallization and data collection: The SEC-purified H078.14UFO²-BG trimer complexes was concentrated to ˜5 mg/ml before subjectedto extensive crystallization trials at both 4° C. and 20° C. Crystalsfor protein complex containing Fab PGT124 and 35022 bound to UFO²-BGtrimer were obtained from 0.1 M calcium acetate, 0.1 M MES (pH 6.0), 15%(v/v) PEG400, and harvested and cryo-protected with 25% glycerol,followed by immediate flash cooling in liquid nitrogen. In currentcondition, the best crystal was diffracted to 6.20 Å resolution and thedata was collected at beamline 12-2 at the Stanford Synchtron RadiationLightsource, processed with HKL-2000, and indexed in space group P63with 99.7% completeness with unit cell parameters a=b=129.3 Å, c=314.5Å.

Structure determination and refinement: The H078.14 UFO²-BGtrimerstructure bound to PGT124 and 35022 were solved by molecular replacement(MR) using Phaser with the one protomer of 35022:BG505 SOSIP.664structure (PDB: SCEZ), and PGT124 Fab structure (PDB: 4R26). Thestructures were refined using Phenix, with Coot used for model buildingand MolProbity for structure validation. Due to the limited resolutionof the datasets, two B-factor groups per residue refinement were used.Furthermore, positional coordinate refinement was enforced using areference model set of restraints. The final R_(cryst) and R_(free)values for complex structure are 25.0% and 31.4%. Figures were generatedwith PyMol and Chimera. In the crystal structure, the residues werenumbered according to the Kabat definition for FAbs and according to theHXBc2 system for gp140.

Negative-stain electron microscopy: UFO²—BG trimers andtrimer-presenting nanoparticles were analyzed by negative-stain EM. A 34aliquot containing ˜0.01 mg ml⁻¹ of the trimers or nanoparticles wasapplied for 15 s onto a carbon-coated 400 Cu mesh grid that had beenglow discharged at 20 mA for 30 s, then negatively stained with 2% (w/v)uranyl formate for 30 s. Data were collected using a FEI Tecnai Spiritelectron microscope operating at 120 kV, with an electron dose of ˜25 e⁻A⁻² and a magnification of 52,000× that resulted in a pixel size of 2.05Å at the specimen plane. Images were acquired with a Tietz 4k×4kTemCam-1⁷416 CMOS camera using a nominal defocus of 1500 nm and theLeginon package. UFO²—BG trimer particles were selected automaticallyfrom the raw micrographs using DoG Picker, while trimer-presentingnanoparticles were selected manually using the Appion Manual Picker.Both were put into particle stack using the Appion software package.Reference-free, two-dimensional (2D) class averages were calculatedusing particles binned by two via iterative multivariate statisticalanalysis (MSA)/multireference alignment (MRA) and sorted into classes.To analyze the quality of the trimers (native-like and non-native), thereference free 2D class averages were examined by eye as previouslydescribed (de Taeye et al., Cell 163, 1702-1715, 2015).

Bio-Layer Interferometry (BLI): The kinetics of trimer and nanoparticlebinding to bNAbs and non-NAbs was measured using an Octet Red96instrument (forteBio, Pall Life Sciences). All assays were performedwith agitation set to 1000 rpm in forteBio 1× kinetic buffer. The finalvolume for all the solutions was 200 μl per well. Assays were performedat 30° C. in solid black 96-well plates (Geiger Bio-One). 5 μg ml⁻¹ ofantibody in 1× kinetic buffer was loaded onto the surface of anti-humanFc Capture Biosensors (AHC) for 300 s. A 60 s biosensor baseline stepwas applied prior to the analysis of the association of the antibody onthe biosensor to the antigen in solution for 200 s. A two-foldconcentration gradient of antigen, starting at 200 nM for trimers and14-35 nM for nanoparticles depending on the size, was used in atitration series of six. The dissociation of the interaction wasfollowed for 300 s. Correction of baseline drift was performed bysubtracting the mean value of shifts recorded for a sensor loaded withantibody but not incubated with antigen and for a sensor withoutantibody but incubated with antigen. Octet data were processed byforteBio's data acquisition software v.8.1. Of note, for apex-directedbNAbs, experimental data were fitted with the binding equationsdescribing a 2:1 interaction to achieve the optimal fitting results.

B cell activation assay: Generation of K46 B-cell lines expressingPGT121, PGT145 or VRCO1 has been previously described (Ota et al., J.Immunol. 189, 4816-48242012). In brief, K46 cells expressing adoxycyclin-inducible form of bNAb B cell receptors (BCRs) weremaintained in advanced DMEM (Gibco), supplemented with 10% FCS,Pen/Strep antibiotics, and 2 μg Puromycin (Gibco). Cells were treatedovernight in 1 μg doxycyclin (Clontech) to induce human BCR expression.After loading with Indo-1 (Molecular Probes) at 1 μM for one hour at 37°C., washed cells were stimulated with the indicated agents at aconcentration of 10 μg ml⁻¹: anti-mouse IgM (Jackson ImmunoResearch);UFO²—BG or an HR1-redesigned gp140 trimer with a T-helper epitope(PADRE) fused to the C-terminus; UFO²—BG-FR or I3-01 nanoparticlepresenting an HR1-redesigned gp140 trimer. Calcium mobilization wasassessed on a LSR II flow cytometer (BD). In each run, the unstimulatedB cells were first recorded for 60 s, with the testing immunogen added,mixed thoroughly, and recorded for 180 s, followed by addition of 1 μlof 1 μg ml⁻¹ ionomycin (Sigma) and recording for another 60 s to verifyindo loading.

Mouse immunization and serum IgG purification: Seven-week-old BALB/cmice were purchased from The Jackson Laboratory. The mice were housed inventilated cages in environmentally controlled rooms at TSRI, incompliance with an approved IACUC protocol and AAALAC guidelines. Atweek 0, each mouse was immunized with 200 μl of antigen/adjuvant mixcontaining 50 μg of antigen and 100 μl AddaVax adjuvant (Invivogen) or50 μl PIKA adjuvant (Yisheng Biopharma) per manufacturer's instructionvia the intraperitoneal (i.p.) route. At week 3 and week 6, the animalswere boosted with 50 μg of antigen formulated in AddaVax or PIKAadjuvant. At week 8, the animals were terminally bled through the retroorbital membrane using heparinized capillary tubes. Samples were dilutedwith an equal volume of PBS and then overlayed on 4.5 ml ofFicoll/Histopaque in a 15 ml SepMate tube (StemCell) and spun at 1200RPM for 10 min at 20° C. to separate plasma and cells. The plasma washeat inactivated at 56° C. for 1 hour, spun at 1200 RPM for 10 min andsterile filtered. The cells were washed once in PBS and then resuspendedin 1 ml of ACK Red Blood Cell lysis buffer (Lonza). After 2 rounds ofwashing with PBS, PBMCs were resuspended in 2 ml of Bambanker FreezingMedia (Lymphotec Inc.). Spleens were also harvested and grounded againsta 40-μm cell strainer (BD Falcon) to release the splenocytes into a cellsuspension. The cells were centrifuged, washed in PBS and then treatedwith 10 ml of RBC lysis buffer as per manufacturer specifications, andresuspended in Bambanker Freezing Media for cell freezing. One-third ofthe total serum per mouse, or 600 μl of serum, was purified using a0.2-ml protein G spin kit (Thermo Scientific) following themanufacturer's instructions. Purified serum IgGs obtained from four micewithin each group were combined for characterization by ELISA and HIV-1neutralization assays.

Enzyme-linked immunosorbent assay (ELISA): Each well of a Costar™96-well assay plate (Corning) was first coated with 50 μl PBS containing0.2 μg of the appropriate antigens. The plates were incubated overnightat 4° C., and then washed five times with wash buffer containing PBS and0.05% (v/v) Tween 20. Each well was then coated with 150 μl of ablocking buffer consisting of PBS, 20 mg ml⁻¹ blotting-grade blocker(Bio-Rad), and 5% (v/v) FBS. The plates were incubated with the blockingbuffer for 1 hour at room temperature, and then washed 5 times with washbuffer. Purified mouse IgGs were diluted in the blocking buffer to amaximum concentration of 100 μg ml⁻¹, followed by a 10-fold dilutionseries. For each antibody dilution, a total of 50 IA volume was added tothe appropriate wells. Each plate was incubated for 1 h at roomtemperature, and then washed 5 times with wash buffer. A 1:2000 dilutionof horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody(Jackson ImmunoResearch Laboratories) was then made in the wash buffer,with 50 μl of this diluted secondary antibody added to each well. Theplates were incubated with the secondary antibody for 1 hr at roomtemperature, and then washed 5 times with wash buffer. Finally, thewells were developed with 50 μl of TMB (Life Sciences) for 3-5 minbefore stopping the reaction with 50 μl of 2 N sulfuric acid. Theresulting plate readouts were measured at a wavelength of 450 nm.

Pseudovirus production and neutralization assays: Pseudoviruses weregenerated by transfection of 293 T cells with an HIV-1 Env expressingplasmid and an Env-deficient genomic backbone plasmid (pSG3AEnv), asdescribed previously. Pseudoviruses were harvested 72 hourspost-transfection for use in neutralization assays. Neutralizingactivity of purified mouse serum IgGs was assessed using a single roundof replication pseudovirus assay and TZM-b1 target cells, as describedpreviously. Briefly, TZM-b1 cells were seeded in a 96-well flat bottomplate. To this plate was added pseudovirus, which was preincubated withserial dilutions of mouse serum IgG for 1 hour at 37° C. Luciferasereporter gene expression was quantified 72 hours after infection uponlysis and addition of Bright-Glo® Luciferase substrate (Promega). Todetermine IC50 values, dose-response curves were fit by nonlinearregression.

Mouse repertoire sequencing and bioinformatics analysis: A 5′-RACEprotocol has been developed for unbiased sequencing of mouse B-cellrepertoires, as previously described. Briefly, RNA (including mRNA) wasextracted from total PBMCs of each mouse into 30 μl of water with RNeasyMini Kit (Qiagen). 5′-RACE was performed with SMARTer RACE cDNAAmplification Kit (ClonTech). The immunoglobulin PCRs were set up withPlatinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a totalvolume of 50 with 5 μl of cDNA as template, 1 μl of 5′-RACE primer, and1 μl of 10 μM reverse primer. The 5′-RACE primer contained a PGM/S5 P1adaptor, while the reverse primer contained a PGM/S5 Å adaptor. Weadapted the mouse 3′-C_(y)1-3 and 3′-C_(μ) inner primers as reverseprimers for 5′-RACE PCR processing of the heavy chains. A total of 25cycles of PCR was performed and the expected PCR products (500-600 bp)were gel purified (Qiagen). NGS was performed on the Ion S5 system.Briefly, heavy chain libraries from the same group were quantitatedusing Qubit® 2.0 Fluorometer with Qubit® dsDNA HS Assay Kit, and thenmixed using a ratio of 1:1:1:1 for sequencing. Template preparation and(Ion 520) chip loading were performed on Ion Chef using the Ion 520/530Ext Kit, followed by sequencing on the Ion S5 system with defaultsettings. The mouse antibodyomics pipeline was used to process the rawdata and to determine the distributions of heavy chain germline geneusage.

Example 7 T-Helper Epitope-Encapsulated Nanoparticles

Although the use of a T-helper epitope as a linker to connect HIV-1gp140 and nanoparticle backbone produced HIV-1 trimer-presentingnanoparticles with desirable antigenic and immunogenic properties (FIGS.1-3), the assembly of such nanoparticles appeared to be affected by thehydrophobic T-helper epitopes exposed on the nanoparticle surface. Toimprove nanoparticle assembly and purity, an alternative strategy forincorporating T-cell helper epitope into nanoparticle vaccine design wasexamined. Instead of inserting a T-helper epitope between antigen andnanoparticle backbone on the outside surface, this T-helper epitope wasgenetically fused to the C-terminus of the nanoparticle subunit via ashort, flexible peptide spacer. The expected outcome would be ananoparticle vaccine with twenty HIV-1 gp140 trimers displayed on theoutside surface and sixty hydrophobic T-helper epitopes encapsulatedinside the nanoparticle shell (FIG. 4A). This design was devised basedon the observations that both E2p and I3-01 are large 60-meric nanocageswith hollow interiors and that almost all proteins prefer a hydrophobiccore and a charged/hydrophilic surface to achieve stability in solution.

We tested this strategy with a pan-reactive T-helper epitope, PADRE. Inthe construct design, the C-terminus of HIV-1 BG505 gp140 was fused tothe N-terminus of the nanoparticle subunit with a 1G spacer (for E2μ) orwith a 10aa GGGGSGGGGS spacer (for I3-01), both of which contained anenzymatic site (AS) preceding the spacer, and then the N-terminus ofPADRE was fused to the C-terminus of the nanoparticle subunit with a 5aaGGGGS spacer. The two resulting fusion constructs were expressedtransiently in 25 ml of ExpiCHO cells and purified using a 2G12 antibodyaffinity column. The obtained protein was analyzed by size-exclusionchromatography (SEC) on a Superose 6 10/300 GL column. For bothconstructs, we observed peaks at 6-7 mL corresponding to well-formednanoparticles (FIG. 4B). Considering the smaller-scale transfection (25ml vs 100 ml in FIG. 1F), the actual nanoparticle yield was notablyimproved compared to the design in which the T-helper epitope is used asa linker outside the nanoparticle. The 2G12-purified nanoparticles werefurther analyzed by negative-stain EM. Fully assembled nanoparticleswith spikes on the surface can be recognized from the raw micrographsderived from negative-stain EM (FIG. 4C). Taken together, SEC and EMdata confirmed that T-helper epitope encapsulation might presents aneffective strategy for designing HIV-1 nanoparticle vaccines withembedded T-cell help.

A T-helper epitope can be fused to the C-terminus of the subunit of aself-assembling nanoparticle via a short peptide spacer. The C-terminusof HIV-1 gp140 can be fused to the N-terminus of the subunit of theabovementioned nanoparticle. When these fusion subunits assemble into ananoparticle, it will present 8 or 24 HIV-1 gp140 trimers on the outsidesurface of the nanoparticle while encapsulating 24 or 60 T-helperepitopes inside the nanoparticle shell. The HIV-1 gp140 trimers on theoutside surface of the nanoparticle will induce an anti-HIV-1 B-cellresponse, while the dense cluster of T-helper epitopes inside thenanoparticle will induce a broadly reactive T-cell response upon thedigestion of the nanoparticle protein.

The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. It isunderstood that various modifications can be made to the presentinvention without departing from the spirit and scope thereof.

It is further noted that all publications, sequence accession numbers,patents and patent applications cited herein are hereby expresslyincorporated by reference in their entirety and for all purposes as ifeach is individually so denoted. Definitions that are contained in textincorporated by reference are excluded to the extent that theycontradict definitions in this disclosure.

What is claimed is:
 1. A HIV-1 vaccine immunogen, comprising an HIV-1Env-derived trimer protein presented on a self-assembling nanoparticle,wherein a linker sequence (a) is fused to the C-terminus of thenanoparticle subunit while the HIV-1 trimer protein subunit is fused tothe N-terminus of the nanoparticle subunit or (b) links the HIV-1 trimerprotein to the N-terminus of the nanoparticle subunit, wherein theself-assembling nanoparticle has a subunit sequence as shown in any oneof SEQ ID NOs:6-17 or a conservatively modified variant thereof.
 2. TheHIV-1 vaccine immunogen of claim 1, wherein the HIV-1 Env-derived trimerprotein is an uncleaved prefusion-optimized (UFO) gp140 trimer.
 3. TheHIV-1 vaccine immunogen of claim 2, wherein the UFO gp140 trimer is achimeric trimer comprising a redesigned gp41_(ECTO) domain from HIV-1strain BG505, wherein the redesigned gp41_(ECTO) domain contains (1) HR1N-terminal bend replaced with a stabilizing loop sequence and (2) acleavage-site linker.
 4. The HIV-1 vaccine immunogen of claim 1, whereinthe linker sequence comprises a T-helper epitope sequence or aglycine-serine linker or both.
 5. The HIV-1 vaccine immunogen of claim1, wherein the linker sequence comprises the sequence as shown in anyone of SEQ ID NOs:1-3, or a conservatively modified variant thereof. 6.The HIV-1 vaccine immunogen of claim 1, wherein the linker sequencecomprises 1 to 5 tandem repeats of GGGGS (SEQ ID NO:4) or GSGSG (SEQ IDNO:19).
 7. The HIV-1 vaccine immunogen of claim 1, wherein the linkersequence is fused to the C-terminus of the nanoparticle subunit via ashort peptide spacer, and a second peptide spacer links the HIV-1 trimerprotein subunit to the N-terminus of the nanoparticle subunit.
 8. Apharmaceutical composition, comprising the HIV-1 vaccine immunogen ofclaim 1, and a pharmaceutically acceptable carrier.
 9. Thepharmaceutical composition of claim 8, further comprising an adjuvant.10. A method of inducing an HIV-1 neutralizing antibody in a subject,comprising administering to the subject a therapeutically effectiveamount of the HIV-1 vaccine immunogen of claim 1, thereby inducing aHIV-1 neutralizing antibody in the subject.
 11. The method of claim 10,wherein HIV-1 vaccine immunogen comprises an UFO gp140 trimer, aself-assembling nanoparticle generated with a subunit sequence as shownin any one of SEQ ID NOs:6-17, and a T-helper epitope sequencecomprising the sequence as shown in SEQ ID NO:1, wherein the T-helperepitope sequence (a) is fused to the C-terminus of the nanoparticlesubunit via a short peptide spacer while the UFO gp140 trimer subunit isfused to the N-terminus of the nanoparticle subunit or (b) covalentlylinks the UFO gp140 trimer subunit at its C-terminus to the N-terminusof the nanoparticle subunit.
 12. The method of claim 11, wherein theT-helper epitope sequence fused to the C-terminus of the nanoparticlesubunit is encapsulated within the nanoparticle upon self-assembly ofthe nanoparticle.
 13. The HIV-1 vaccine immunogen of claim 1, whereinthe self-assembling nanoparticle comprises a trimeric sequence.
 14. TheHIV-1 vaccine immunogen of claim 1, wherein the HIV-1 Env-derived trimerprotein is gp140.
 15. The HIV-1 vaccine immunogen of claim 1, whereinthe HIV-1 Env-derived trimer protein is an uncleaved prefusion-optimized(UFO) gp140 trimer.
 16. The HIV-1 vaccine immunogen of claim 15, whereinthe UFO gp140 trimer is a chimeric trimer comprising a redesignedgp41_(ECTO) domain from HIV-1 strain BG505, wherein the redesignedgp41_(ECTO) domain contains (1) HR1 N-terminal bend replaced with astabilizing loop sequence and (2) a cleavage-site linker.
 17. The HIV-1vaccine immunogen of claim 15, wherein the HIV-1 Env-derived trimer isan UFO gp140 trimer, the self-assembling nanoparticle is generated witha subunit sequence as shown in any one of SEQ ID NOs:17, and the linkersequence comprises the sequence as shown in SEQ ID NO:1.
 18. The HIV-1vaccine immunogen of claim 7, wherein the linker sequence isencapsulated within the nanoparticle.
 19. The method of claim 10,wherein the HIV-1 Env-derived trimer protein is an uncleavedprefusion-optimized (UFO) gp140 trimer.
 20. The method of claim 19,wherein the UFO gp140 trimer is a chimeric trimer comprising aredesigned gp41_(ECTO) domain from HIV-1 strain BG505, wherein theredesigned gp41_(ECTO) domain contains (1) HR1 N-terminal bend replacedwith a stabilizing loop sequence and (2) a cleavage-site linker.